Project Summary We have shown that purified hematopoietic stem cells (HSC), first isolated by us, can be allotransplanted without GvH (they lack T cells), block autoimmune type 1 diabetes (and Systemic Lupus Erythematosus), and induce transplant tolerance to HSC donor tissues/organs. However, purified HSC transplantation is not used, in part due to the toxicity of the conditioning regimens. Therefore, it is vital that we improve our understanding of how HSCs interact with their local supporting environment?the hematopoietic niche. The goal of this research is to further identify and characterize the cellular and molecular components of the bone marrow (BM) HSC niche, and to understand how the ongoing inter-cellular communication between HSCs and their niche regulates their homing, anchoring, survival and function in health and in blood diseases. In previous versions of this grant we established antibodies in place of toxic regimens to condition recipients and will further modify the regimens to improve engraftment and accelerate reconstitution. Further, understanding the HSC-niche interaction has implications for stem cell competitions we have elucidated in pathology, for example, in hematopoietic disorders such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), and expansion of mutated clones to give clonal hematopoiesis, some that give rise to acute myeloid leukemia (AML). In the run-up to leukemia and MDS, clones of HSCs with pre-cancer mutations outcompete normal HSCs, but the total number of HSCs appears not to change, implying control of HSC numbers by the niche. The natural recirculation of HSCs allows highly competitive clones to dominate HSC niches throughout the body, so what we learn about normal HSC development and homing will also likely apply to a variety of disorders of hematopoiesis, including clonal hematopoiesis and leukemias. The experiments here concern HSC homing, anchoring, support, and niche competition. In Aim 1, we will utilize long-term (LT)-HSC reporter mice to explore the specificity of the niche, identify the cell types that form direct contact with LT-HSC, and analyze the adhesion molecules, cytokines and chemokines responsible for HSC and niche cross-talk. In Aim 2 we propose to understand the role of HSC-surrounding BM cells?focusing on adjacent sinusoidal endothelial cells and the essential BM stromal subsets and their expressed factors. That builds on our discovery and characterization of the skeletal stem cells (SSC), which clonally generate bone, cartilage, and several distinct BM stromal cells that support hematopoiesis. We plan to produce mice in which each cell subset in the niche, including HSC, can be engineered to knock out identified genes to decipher the complexity of niche-HSC interactions. This could lead to an understanding if there are means to increase the number of functional niches, whether modulation of the molecular interactions could clear the niche specifically to promote the HSC engraftment, and to provide targets for modulating clonal HSC expansions. !